The half maximal inhibitory concentration (IC50) of FG4592 on KDM6A/UTX or MAPK p38α was measured by the ChemPartner company. The IC50 of FG4592 on JMJD3 was measured by Huawei Pharmaceutical company. Briefly, FG4592 was transferred to the assay plate with serial dilution. FG4592 with different concentrations was added to the substrate solution in a 384-well plate. After incubating and adding the stop solution, the endpoint of the plates of KDM6A/UTX or MAPK p38α was read with EnSpire with Alpha mode or Caliper. The IC50 of FG4592 on JMJD3 was measured by Huawei Pharmaceutical Com. Ltd. All of the enzymatic reactions were conducted in the mixture containing assay buffer, histone H3 peptide substrate, demethylase enzyme, and FG4592 with different concentrations. After enzymatic reactions and adding anti-Mouse Acceptor beads and primary antibody, the samples were measured in an AlphaScreen microplate reader (EnSpire Alpha 2390 Multilabel Reader, PerkinElmer). Enzyme activity assays were performed in duplicates at each concentration, and the A-screen intensity data were analyzed and compared. Brief methods and reports can be provided if needed.
Enspire alpha 2390 multilabel reader
Manufactured by PerkinElmer
The EnSpire Alpha 2390 Multilabel Reader is a versatile laboratory instrument designed for a wide range of microplate-based assays. It features high-performance optics and a flexible configuration to support various detection modes, including absorbance, fluorescence, and luminescence measurements.
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Lab products found in correlation
2 protocols using enspire alpha 2390 multilabel reader
1
Inhibition of KDM6A/UTX, MAPK p38α, and JMJD3 by FG4592
2
Enzymatic Demethylase Activity Assay
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All
of the enzymatic reactions were conducted in duplicate at RT for 60
min in a 10 μL mixture containing assay buffer, histone H3 peptide
substrate, demethylase enzyme, and the test compound. These 10 μL
reactions were carried out in wells of 384-well Optiplate (PerkinElmer).
After enzymatic reactions, 5 μL of anti-Mouse Acceptor beads
(PerkinElmer, diluted 1:250 with 1× detection buffer and 5 μL
of primary antibody (BPS, diluted 1:100 with 1× detection buffer)
were added to the reaction mix. After brief shaking, the plate was
incubated for 30 min. Finally, 10 μL of AlphaScreen Streptavidin-conjugated
donor beads (PerkinElmer, diluted 1:125 with 1× detection buffer)
were added. In 15 min, the samples were measured in AlphaScreen microplate
reader (EnSpire Alpha 2390 Multilabel Reader, PerkinElmer). Enzyme
activity assays were performed in duplicates at each concentration.
The AlphaScreen intensity data were analyzed and compared. In the
absence of the compound, the intensity (Ce) in each data set was defined as 100% activity. In the absence of
enzyme, the intensity (C0) in each data
set was defined as 0% activity. The percent activity in the presence
of each compound was calculated according to the following equation:
% activity = (C – C0)/(Ce – C0), where C is the A-screen intensity in the
presence of the compound.
of the enzymatic reactions were conducted in duplicate at RT for 60
min in a 10 μL mixture containing assay buffer, histone H3 peptide
substrate, demethylase enzyme, and the test compound. These 10 μL
reactions were carried out in wells of 384-well Optiplate (PerkinElmer).
After enzymatic reactions, 5 μL of anti-Mouse Acceptor beads
(PerkinElmer, diluted 1:250 with 1× detection buffer and 5 μL
of primary antibody (BPS, diluted 1:100 with 1× detection buffer)
were added to the reaction mix. After brief shaking, the plate was
incubated for 30 min. Finally, 10 μL of AlphaScreen Streptavidin-conjugated
donor beads (PerkinElmer, diluted 1:125 with 1× detection buffer)
were added. In 15 min, the samples were measured in AlphaScreen microplate
reader (EnSpire Alpha 2390 Multilabel Reader, PerkinElmer). Enzyme
activity assays were performed in duplicates at each concentration.
The AlphaScreen intensity data were analyzed and compared. In the
absence of the compound, the intensity (Ce) in each data set was defined as 100% activity. In the absence of
enzyme, the intensity (C0) in each data
set was defined as 0% activity. The percent activity in the presence
of each compound was calculated according to the following equation:
% activity = (C – C0)/(Ce – C0), where C is the A-screen intensity in the
presence of the compound.
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